Chronic Exposure to Combined Carcinogens Enhances Breast Cell Carcinogenesis with Mesenchymal and Stem-Like Cell Properties

Breast cancer is the most common type of cancer affecting women in North America and Europe. More than 85% of breast cancers are sporadic and attributable to long-term exposure to small quantities of multiple carcinogens. To understand how multiple carcinogens act together to induce cellular carcinogenesis, we studied the activity of environmental carcinogens 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and benzo[a]pyrene (B[a]P), and dietary carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) using our breast cell carcinogenesis model. Our study revealed, for the first time, that combined NNK and B[a]P enhanced breast cell carcinogenesis chronically induced by PhIP in both non-cancerous and cancerous breast cells. Co-exposure was more potent than sequential exposure to combined NNK and B[a]P followed by PhIP in inducing carcinogenesis. Initiation of carcinogenesis was measured by transient endpoints induced in a single exposure, while progression of carcinogenesis was measured by acquisition of constitutive endpoints in cumulative exposures. Transient endpoints included DNA damage, Ras-Erk-Nox pathway activation, reactive oxygen species elevation, and increased cellular proliferation. Constitutive endpoints included various cancer-associated properties and signaling modulators, as well as enrichment of cancer stem-like cell population and activation of the epithelial-to-mesenchymal transition program. Using transient and constitutive endpoints as targets, we detected that a combination of the green tea catechins ECG and EGCG, at non-cytotoxic levels, was more effective than individual agents in intervention of cellular carcinogenesis induced by combined NNK, B[a]P, and PhIP. Thus, use of combined ECG and EGCG should be seriously considered for early intervention of breast cell carcinogenesis associated with long-term exposure to environmental and dietary carcinogens

Efficient identification of tetR-expressing cell lines for tetracycline-regulated gene expression

The technology of tetracycline-inducible gene expression has been successfully used in experimental biology to identify the function and downstream signaling pathways of an interested gene. It has been significantly improved to meet the criteria with specificity to exogenous non-toxic inducers, independent regulation from cellular pathways, and dose-dependent inducibility and reversibility. However, to establish tetracycline-inducible gene expression in mammalian cells is still a time-and effort-consuming process. With a tetracycline-inducible gene expression system T-REx, we have developed a practical protocol to use the oncogenic H-ras gene as a dominant reporter gene to increase efficiency in attaining desired cell lines in which ectopic expression of a particular gene in cells can be introduced and reversibly induced by the presence or absence of tetracycline in cultures

Efficient identification of tetR-expressing cell lines for tetracycline-regulated gene expression

The technology of tetracycline-inducible gene expression has been successfully used in experimental biology to identify the function and downstream signaling pathways of an interested gene. It has been significantly improved to meet the criteria with specificity to exogenous non-toxic inducers, independent regulation from cellular pathways, and dose-dependent inducibility and reversibility. However, to establish tetracycline-inducible gene expression in mammalian cells is still a time-and effort-consuming process. With a tetracycline-inducible gene expression system T-REx, we have developed a practical protocol to use the oncogenic H-ras gene as a dominant reporter gene to increase efficiency in attaining desired cell lines in which ectopic expression of a particular gene in cells can be introduced and reversibly induced by the presence or absence of tetracycline in cultures

Intervention of NBP-induced carcinogenesis.

<p>(A-1 to A-4) MCF10A cells were treated with NBP in the absence and presence of ECG (E), EGCG (G), or a combination of ECG and EGCG (E+G) for 24 h. (B-1 to B-10) MCF10A (10A) cells were exposed to NBP in the absence and presence of 20 µg/mL ECG, 20 µg/mL EGCG, or combined 10 µg/mL ECG and 10 µg/mL EGCG (E/G) for 10 cycles, resulting in the NBP10, NBP-E10, NBP-G10, and NBP-E/G10 cell lines, respectively. (A-1 and B-6) Relative level of ROS as fold induction (X, arbitrary unit) was normalized by the level determined in untreated cells, set as 1. (A-2) Relative DNA damage was measured by a comet assay and normalized by the value of average tail moment determined in untreated counterpart cells, set as 1 (X, arbitrary unit). (A-3 and B-7) Cell lysates were analyzed by immunoblotting using specific antibodies to detect levels of H-Ras, phosphorylated-Erk1/2 (p-Erk1/2), Erk1/2, and Nox-1, with β-actin as a control, and these levels were quantified by densitometry. Levels of H-Ras and Nox-1 were calculated by normalizing with the level of β-actin and the level set in untreated control cells as 1 (X, arbitrary unit). Levels of specific phosphorylation of Erk1/2 (p/Erk) were calculated by normalizing the levels of p-Erk1/2 with the levels of Erk1/2, then the level set in control cells as 1 (X, arbitrary unit). (A-4 and B-5) Relative cell proliferation was determined and normalized by the value of BrdU detected in untreated cells, set as 100%. (B-1) To determine cellular acquisition of RDGF, cells were maintained in LM medium for 10 days. Cell colonies ≥0.5 mm diameter were counted. (B-2) To determine cellular acquisition of AIG, cells were seeded in soft agar for 14 days. Cell colonies ≥0.1 mm diameter were counted. (B-3) Cellular migratory and (B-4) invasive activities were determined by counting the numbers of cells translocated through a polycarbonate filter without or with coated Matrigel, respectively, in 10 arbitrary visual fields. (B-8) To determine cellular acquisition of the ability of serum-independent non-adherent growth (SINAG), cells were seeded in non-adherent cultures for 10 days; then, mammospheres (≥0.1 mm diameter) were counted. (B-9) Mammospheres were collected and trypsinized, and ALDH-expressing (ALDH⁺) cell population (%) was measured by flow cytometry. (B-10) Cell lysates were analyzed by immunoblotting using specific antibodies to detect levels of EpCAM, E-cadherin, MMP-9 and Vimentin, with β-actin as a control, and these levels were quantified by densitometry. The levels of EpCAM, E-cadherin, MMP-9 and Vimentin were calculated by normalizing with the level of β-actin and the level set in untreated control cells as 1 (X, arbitrary unit). Columns, mean of triplicates; bars, SD. All results are representative of three independent experiments. Statistical significance is indicated by * <i>P</i><0.05, ** <i>P</i><0.01, and *** <i>P</i><0.001.</p

Enhanced cellular acquisition of cancer-associated properties by combined carcinogens.

<p>(A) MCF10A cells were treated with combined 100 pmol/L NNK and 100 pmol/L B[a]P (NB), 10 nmol/L PhIP (P), or combined NB and PhIP (NBP) in the absence and presence of 10 µmol/L U0126 (U0) or 5 mmol/L NAC for 24 h. DNA damage was measured by a comet assay and normalized by the value of average tail moment determined in untreated counterpart cells, set as 1 (X, arbitrary unit). Representative images detected in the comet assay are shown. (B-1 to B-6) MCF10A (10A) cells were repeatedly exposed to NB, PhIP, or NBP for 20 cycles, resulting in the NB20, P20, and NBP20 cell lines, respectively. NB20 cells were then exposed to PhIP for an additional 20 cycles resulting in the NB20/P20 cell line. MCF10A-Ras (Ras) cells were used as a malignant control. (C-1 to C-3) NB20/P20 and NBP20 cells were treated with 10 µmol/L U0 or 5 mmol/L NAC for 48 h. (B-1) To determine cellular acquisition of RDGF, cells were maintained in LM medium for 10 days. Cell colonies ≥0.5 mm diameter were counted. (B-2) To determine cellular acquisition of AIG, cells were seeded in soft agar for 14 days. Cell colonies ≥0.1 mm diameter were counted. (B-3 and C-2) Relative level of ROS as fold induction (X, arbitrary unit) was normalized by the level determined in untreated cells, set as 1. (B-4 and C-3) Relative cell proliferation was determined and normalized by the value of BrdU detected in untreated cells, set as 100%. (B-5) Total RNA was isolated and analyzed by RT-PCR with specific primers to determine relative gene expression levels of H-Ras, with β-actin as a control, and these levels were quantified by densitometry. (B-6 and C-1) Cell lysates were analyzed by immunoblotting using specific antibodies to detect levels of H-Ras, phosphorylated-Erk1/2 (p-Erk1/2), Erk1/2, and Nox-1, with β-actin as a control, and these levels were quantified by densitometry. The levels of H-Ras (Ras/actin) and Nox-1 (Nox/actin) were calculated by normalizing with the level of β-actin and the level set in untreated control cells as 1 (X, arbitrary unit). Levels of specific phosphorylation of Erk1/2 (p/Erk) were calculated by normalizing the levels of p-Erk1/2 with the levels of Erk1/2, then the level set in control cells as 1 (X, arbitrary unit). Columns, mean of triplicates; bars, SD. All results are representative of three independent experiments. Statistical significance is indicated by * <i>P</i><0.05, ** <i>P</i><0.01, and *** <i>P</i><0.001.</p

Enhanced acquisition of mesenchymal and stem-like cell properties by combined carcinogens.

<p>(A) To determine cellular acquisition of the ability of serum-independent non-adherent growth (SINAG), MCF10A (10A), NB20, P20, NB20/P20, NBP20, and MCF10A-Ras (Ras) cells were seeded in non-adherent cultures for 10 days; then, mammospheres (≥0.1 mm diameter) were counted. (B) Mammospheres were collected and trypsinized, and ALDH-expressing (ALDH⁺) cell population (%) was measured by flow cytometry. (C) Cell lysates were analyzed by immunoblotting using specific antibodies to detect levels of EpCAM, E-cadherin, MMP-9 and Vimentin, with β-actin as a control, and these levels were quantified by densitometry. The levels of EpCAM, E-cadherin, MMP-9, and Vimentin were calculated by normalizing with the level of β-actin and the level set in untreated control cells as 1 (X, arbitrary unit). (D) Cellular migratory and (E) invasive activities were determined by counting the numbers of cells translocated through a polycarbonate filter without or with coated Matrigel, respectively, in 10 arbitrary visual fields. (F-1) Cellular acquisition of increased motility was determined by wound healing assay. The wounded areas were examined (magnification, 100×) 6, 12, and 24 h afterward. Arrows indicate width of wounded areas. (F-2) To quantitatively measure cell motility detected in F-1, the area not healed by the cells was subtracted from the total area of the initial wound to calculate the wound healing area (%) at intervals of 6 (white columns) and 12 h (gray columns). Columns, mean of triplicates; bars, SD. All results are representative of three independent experiments. Statistical significance is indicated by * <i>P</i><0.05, ** <i>P</i><0.01, and *** <i>P</i><0.001.</p

Broad-spectrum antiviral agents

Development of highly effective, broad-spectrum antiviral agents is the major objective shared by the fields of virology and pharmaceutics. Antiviral drug development has focused on targeting viral entry and replication, as well as modulating cellular defense system. High throughput screening of molecules, genetic engineering of peptides, and functional screening of agents have identified promising candidates for development of optimal broad-spectrum antiviral agents to intervene in viral infection and control viral epidemics. This review discusses current knowledge, prospective applications, opportunities, and challenges in the development of broad-spectrum antiviral agents

SOCS3 control the activity of NF-KB induced by HSP70 via degradation of MyD88-adapter-like protein (Mal) in IPEC-J2 cells

Hyperthermia in pigs induces suppressor of cytokine signaling (SOCS) 3 and SOCS4 expression in intestinal gut and causes disruption of inflammation cytokine production. These changes may affect the development of inflammatory bowel disease in heat-stressed pigs. However, the mechanisms are not well understood. Accordingly, in this study, we examined the roles of SOCS members in regulation of the nuclear factor (NF)-KB pathway and heat shock protein (HSP) 70-mediated cytokine induction in 293T human embryonic kidney cells and IPEC-J2 porcine small intestinal epithelial cells. Ectopic expression of HSP70 significantly modulated NF-KB activity (p ≤.05). Moreover, co-expression of SOCS3 or SOCS4 with HSP70 reduced NF-kB activity, which was abolished by SOCS3 or SOCS4 knockdown with short hairpin RNA. Interestingly, MyD88-adaptor-like (Mal) protein was downregulated in cells expressing SOCS3 but not in cells expressing SOCS4. In addition, SOCS3 but not SOCS4 negatively regulated the activity of NF-KB induced by HSP70 overexpression via degradation of Mal. These findings may facilitate the development of novel SOCS3-based therapeutic strategies to control heat stress-related disorders in pigs

Structure-Based Identification of Novel Histone Deacetylase 4 (HDAC4) Inhibitors

Histone deacetylases (HDACs) are important cancer drug targets. Existing FDA-approved drugs target the catalytic pocket of HDACs, which is conserved across subfamilies (classes) of HDAC. However, engineering specificity is an important goal. Herein, we use molecular modeling approaches to identify and target potential novel pockets specific to Class IIA HDAC-HDAC4 at the interface between HDAC4 and the transcriptional corepressor component protein NCoR. These pockets were screened using an ensemble docking approach combined with consensus scoring to identify compounds with a different binding mechanism than the currently known HDAC modulators. Binding was compared in experimental assays between HDAC4 and HDAC3, which belong to a different family of HDACs. HDAC4 was significantly inhibited by compound 88402 but not HDAC3. Two other compounds (67436 and 134199) had IC50 values in the low micromolar range for both HDACs, which is comparable to the known inhibitor of HDAC4, SAHA (Vorinostat). However, both of these compounds were significantly weaker inhibitors of HDAC3 than SAHA and thus more selective, albeit to a limited extent. Five compounds exhibited activity on human breast carcinoma and/or urothelial carcinoma cell lines. The present result suggests potential mechanistic and chemical approaches for developing selective HDAC4 modulators

Cytoplasmic Translocation of Nucleolar Protein NOP53 Promotes Viral Replication by Suppressing Host Defense

NOP53 is a tumor suppressor protein located in the nucleolus and is translocated to the cytoplasm during infection by vesicular stomatitis virus (VSV) and herpes simplex virus type 1 (HSV-1), as shown in our previous study. Cytoplasmic NOP53 interacts with the retinoic acid-inducible gene I (RIG-I) to remove its K63-linked ubiquitination, leading to attenuation of type I interferon IFN-&beta;. In the present study, we found no obvious translocation of NOP53 in infection by a mutant virus lacking ICP4 (HSV-1/d120, replication inadequate). Blocking cytoplasmic translocation of NOP53 by the deletion of its nuclear export sequence (NES) abrogated its ability to support viral replication. These results demonstrated that NOP53 redistribution is related to viral replication. It is interesting that treatment with poly (I:C) or RIG-I-N (a constitutively-active variant) directly induced NOP53 cytoplasmic translocation. To better assess the function of cytoplasmic NOP53 in viral replication, the NOP53-derived protein N3-T, which contains a human immunodeficiency virus (HIV)-derived cell-penetrating Tat peptide at the C-terminal region of N3 (residues 330&ndash;432), was constructed and expressed. The recombinant N3-T protein formed trimers, attenuated the expression of IFN-&beta; and IFN-stimulated genes, as well as decreased the phosphorylation level of interferon regulatory factor 3 (IRF3). Furthermore, N3-T promoted the efficient replication of enveloped and non-enveloped DNA and RNA viruses belonging to 5 families. Our findings expand the understanding of the mechanism by which viruses utilize the nucleolar protein NOP53 for optimal viral replication